Comparison of the structure of turtle pancreatic ribonuclease with those of mammalian ribonucleases

Beintema, Jaap J.; Laan, J.M. van der

doi:10.1016/0014-5793(86)80113-2

Cited by 12 publications

(6 citation statements)

References 19 publications

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“…Interestingly, the Cys65–Cys72 disulfide bond is the only disulfide bond that is not absolutely conserved throughout the ribonuclease A superfamily [5]. For example, this disulfide bond is absent from the RNase A homologs in snapping turtle [47] and iguana [48] as well as from the angiogenins [49,50] and Onconase™[51]. The ribonucleolytic activity of each of these enzymes is less than that of RNase A [48,52–55], as expected from our analysis of catalysis by the C65A/C72A variant (see below).…”

Section: Discussionmentioning

confidence: 99%

Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A

Klink¹,

Woycechowsky²,

Taylor

et al. 2000

European Journal of Biochemistry

138

127

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Disulfide bonds between the side chains of cysteine residues are the only common crosslinks in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124-residue enzyme that contains four interweaving disulfide bonds (Cys26±Cys84, Cys40±Cys95, Cys58±Cys110, and Cys65±Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the conformational stability and catalytic activity of RNase A has been determined by using variants in which each cystine is replaced independently with a pair of alanine residues. Thermal unfolding experiments monitored by ultraviolet spectroscopy and differential scanning calorimetry reveal that wild-type RNase A and each disulfide variant unfold in a two-state process and that each disulfide bond contributes substantially to conformational stability. The two terminal disulfide bonds in the amino-acid sequence (Cys26±Cys84 and Cys58±Cys110) enhance stability more than do the two embedded ones (Cys40±Cys95 and Cys65±Cys72). Removing either one of the terminal disulfide bonds liberates a similar number of residues and has a similar effect on conformational stability, decreasing the midpoint of the thermal transition by almost 40 8C. The disulfide variants catalyze the cleavage of poly(cytidylic acid) with values of k cat /K m that are 2-to 40-fold less than that of wild-type RNase A. The two embedded disulfide bonds, which are least important to conformational stability, are most important to catalytic activity. These embedded disulfide bonds likely contribute to the proper alignment of residues (such as Lys41 and Lys66) that are necessary for efficient catalysis of RNA cleavage.Keywords: conformational stability; differential scanning calorimetry; disulfide bond; enzyme; ribonuclease A.A polypeptide chain can adopt many conformations. Yet, the sequence of its amino-acid residues directs folding to a particular native state [1]. The loss of conformational entropy associated with folding destabilizes the native conformation. This destabilization is overcome by the hydrophobic effect, hydrogen bonds, other noncovalent interactions, and (for many proteins) disulfide bonds [2].Bovine pancreatic ribonuclease A (RNase A; EC 3.1.27.5 [3,4]) provides a superb template with which to dissect the contribution of disulfide bonds to conformational stability. RNase A consists of 124 amino-acid residues and contains four intrachain disulfide bonds (Cys26±Cys84, Cys40±Cys95, Cys58±Cys110, and Cys65±Cys72; Fig. 1). The four disulfide bonds are conserved in all 40 of the known sequences of homologous mammalian pancreatic ribonucleases [5]. Two disulfide bonds (Cys40±Cys95 and Cys65±Cys72) link together surface loops, and two link an a-helix to a b-sheet in the protein core (Cys26±Cys84 and Cys58±Cys110). Three disulfide bonds enclose a loop of similar size (Cys26±Cys84, Cys40±Cys95 and Cys58±Cys110; h 59, 56, and 53, respectively), and the other disulfide bond (Cys65±Cys72; h 8) encloses a smaller loop. In previous work, the cystines of RNase A were replaced with pairs of serine ...

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Section: Discussionmentioning

confidence: 99%

Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A

Klink¹,

Woycechowsky²,

Taylor

et al. 2000

European Journal of Biochemistry

138

127

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“…In Figure 2 we align the sequence of human nonsecretory ribonuclease with the sequences of human pancreatic ribonuclease (Beintema et al, 1984), turtle pancreatic ribonuclease (Beintema et al, 1985b), and human angiogenin (Strydom et al, 1985). If one deletion and three insertions are introduced at external loops of the structure of mammalian pancreatic ribonucleases, a perfect alignment is obtained not only for the active-site residues His-12, His-119, and Lys-411 but also for other residues near the active-site and important for substrate binding including and Asp-121 (Blackburn & Moore, 1982;Beintema & Van der Laan, 1986). Similarly, residues important for formation and conservation of the three-dimensional structure, including the eight half-cystine residues, 1 In the rest of the Discussion, residues are numbered according to the residue numbers of human and bovine pancreatic ribonucleases, except if specified otherwise.…”

Section: Discussionmentioning

confidence: 99%

Amino acid sequence of the nonsecretory ribonuclease of human urine

Beintema¹,

Hofsteenge²,

Iwama³

et al. 1988

Biochemistry

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The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…This information mostly relates to the pancreatic RNases; sequences from more than 40 species are known (Beintema and van der Laan, 1986). Recent investigations and discoveries have suggested that these enzymes are but one subdivision of a much broader superfamily of structurally related proteins, some of which have important biochemical and physiological functions other than RNA digestion.…”

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confidence: 99%

The amino acid sequence of human ribonuclease 4, a highly conserved ribonuclease that cleaves specifically on the 3′ side of uridine

Zhou

Strydom

1993

European Journal of Biochemistry

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. (1986a) Biochemistry 25,7255-72641 was purified from human plasma and its amino acid sequence was determined. This protein is a 119-residue single-chain polypeptidc cross-linked by four disulfide bonds and has an amino-terminal pyroglutaminyl residue. No post-translational modifications were observed during extensive sequence studies on peptide fragments, except for the amino-terminal pyroglutamic acid and a possible deamidation of Asn66. The protein is homologous to the pancreatic ribonucleases and angiogenin, but differs substantially from both of these proteins; the protein sequence has 43% identity with human pancreatic ribonuclease and 39% identity with human angiogenin, as compared to 35% identity between human angiogenin and pancreatic ribonuclease. It is referred to as RNase 4, based on the nomenclature currently used for the genes of pancreatic RNase (RNase 1) and the eosinophil-derived RNases (RNase 2 and RNase 3).Virtually all of the RNasc active-site componcnts, including the catalytic residues Hisl2, His1 19 and Lys41, are preserved. However, some invariant residues of RNase 1 arc replaced, e.g. Lys7 by arginine, Asp14 by histidine, and Pro42 by arginine. RNase 4 contains a unique two-residue deletion at the position corresponding to amino acids 77 and 78 of pancreatic RNase, and its carboxyterminal sequence is truncated at position 122. The deletion in angiogenin at position 21 is also found in RNase 4.RNase 4 is very similar to two RNases isolated from bovine and porcine liver, and together they form a new fanlily in the RNase superfamily. The degree of inter-species similarity (90%) is much greater than within the pancreatic RNase and angiogenin families, which suggests that this ribonuclease could possess a physiologically important function other than general RNA catabolism.The ribonucleases (RNases) constitute a group of enzymes for which a large amount of chemical information now exists. This information mostly relates to the pancreatic RNases; sequences from more than 40 species are known (Beintema and van der Laan, 1986). Recent investigations and discoveries have suggested that these enzymes are but one subdivision of a much broader superfamily of structurally related proteins, some of which have important biochemical and physiological functions other than RNA digestion.One homologue of pancreatic RNase that has an unusual ribonucleolytic activity is angiogenin (Shapiro et al., 1986b). This protein potently elicits neovascularization (Fett et al., 1985; Strydom et al., 1985), induccs second-messenger responses in cultured endothelial cells (Bicknell and Vallee, Con-rsyondeirce ro D. J. Strydom,

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Comparison of the structure of turtle pancreatic ribonuclease with those of mammalian ribonucleases

Cited by 12 publications

References 19 publications

Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A

Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A

Amino acid sequence of the nonsecretory ribonuclease of human urine

The amino acid sequence of human ribonuclease 4, a highly conserved ribonuclease that cleaves specifically on the 3′ side of uridine

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